Production of penicillin in cottonseed meal medium



Patented Sept. 7, 1948 PRODUCTION OF PENICILLTN IN COTTON-- SEED MEAL DIEDIUM Jackson W. Foster, New York, N. Y., and Lloyd E. McDaniel, Rahway, N. J., assignors to Merck & 00., Inc., Rahway, J., a corporation of New Jersey No Drawing. Application January 22, 1946, Serial No. 642,748I

1 12 Claims.

This invention relates to a new and improved process for the production of antibiotics. More particularly it is concerned with a new and improved procedure, utilizing a novel nutritive strain. The invention herein disclosed is directed to an improved process forthe production of penicillin by submerged growth of a penicillin producing strain of Penicillium utilizing, as an ingredient of the nutrient medium, one of the materials disclosed in our copending application as suitable, i. e., cottonseed meal. I

Throughout this application where the term penicillin is used, it is intended to define that anti-bacterial substance produced by molds belonging to the genus Penicillium when the pH of the medium is near the neutral point or on the alkaline side, the properties of which substance have been described by Abraham et a1. (Lancet, 241, 177-89, 1941; and Br. Jr. Exp. Path., 23, 103- 120, 1942) as distinguished from the different principle isolated and described by Coulthard et al. (Nature, 150, 634-35, 1942); and Roberts (Jr. Biol. Chem., 147, 47-58, 1943), and which is formed by molds belonging to the genus Penicillium when the medium remains fairly acid, in the range of approximately pH 3.0 to 5.0.

The processes hitherto known for the produc-.- tion of penicillin, other than that disclosed in our above-identified copending application, involve the stationary surface-pad type of growth of Penicillium notatum in shallow layers of media. Such surface-pad stationary processes are disadvantageous for large-scale commercial operations.

We have now discovered a method whereby penicillin is produced by penicillin-producing strains of Penicillium, the microorganism being propagated under submerged conditions in a nutritive medium which contains cottonseed meal. In addition to cottonseed meal there may be present in the nutrient medium anutrient mineral salt and a source of carbon and energy assimilable by penicillin-producing strains of Pencillium including carbohydrates, glycerol, and mixtures of the same. An agent to stimulate penicillin formation, such as phenylacetylethanolamine, may also be present.

In producing penicillin with our improved nutritive medium under submerged conditions, employing aeration, or aeration together with mechanical agitation, the pH of the aqueous medium in which the penicillin-producing organism is propagated is maintained under neutral or alkaline conditions, i. e., pH values falling within the range 6.0 to 8.5. We have found that under such pH conditions, the microorganism being grow submerged, there is achieved a luxuriant growth of the mold much more rapidly than in cultures of the stationary surface type. Moreover, the organism grows homogeneously throughout the body of the liquid medium, in the form of small discrete colonies, and larger or smaller pieces of vegetative mycelium. The rate of penicillin formation is markedly accelerated under submerged conditions utilizing our improved nutrient medium, and maximum penicillin formation is attained in a shorter period of time than is required for stationary surface growth conditions (from 2 to 6 days as compared with the 7 to 14 days, or longer, required under stationary surface growth conditions).

The aeration, or aeration and agitation, utilized during the propagation of the penicillinproducing microorganism, in accordance with our process, can be secured by means of any method or device, or combinations of devices, which ensure adequate solution of air throughout the medium. For example, air may be passed through the depth of medium in a deep tank,

having been introduced into the tank througha simple pipe opening. Adequate solution of air may be secured by mechanical agitation, for example, by propellers or a turbo-mixer, or the solution of air can be achieved by passing it through a 'sparger or similar device which dis- We have found that the use of cottonseed meal as a nutrient medium for the submerged growth of penicillin-producing organisms utilizing aeration, or aeration and agitation, results in several important advantages in our improved process. First the pH of the cottonseed meal broth containing. added nutrients in which the" penicillin-producing organism is grown tends to' cottonseed meal have been tried out and all have remain fairly constant over the entire period of penicillin production. This is to-be contrasted wlthother available nutrient media, such as the 'medium containing com steep liquor, which has a definite tendency to increase in alkalinity during fermentation, to a pH of 8-8.5, or even higher. Penicillin is less stable under alkaline conditions and, with nutrient media other than cottonseed meal, this tendency to increase in alkalinity during fermentation has necessitated reducing the growing period, thereby producing a nutrient medium of lowered penicillin content. By utilizin cottonseedmeal as the primary constituent of the nutrient medium greater uniformity in pH during the growing period is maintained, thereby avoiding the development of high pH values during fermentation. It is therefore possible to prolong the growing period, thereby securing maximum penicillin content in the nutrient medium.

Another advantage of the cottonseed meal medium over other available nutrient media, such, for example, as corn steep liquor, is that there is but little foaming of the cottonseed meal medium under production conditions. With some media, such, for example, as media containing .corn steep liquor as the primary nutrient, foaming becomes so marked as to require reducing the air supply during fermentation. This results in reduced-growth and lessened penicillin content in the nutrient medium. If foaming-over occurs contamination'results, and the process utilizing cottonseed meal as a nutrient medium possesses distinct advantages in that the tendency toward foaming is very much reduced. I

We have found, moreover, that the penicillinproducing organism grows more rapidly in the new nutrient medium utilizing cottonseed meal as its essential element than it does in other available nutrient media, such as the usual corn steep liquor medium utilized to some extent in commercial production. The broth has a higher penicillin potency per unit of volume than with other nutrient media. Moreover, the activity of the extracted penicillin is usually higher when cottonseed meal has been utilized in the nutrient medium than is characteristic of penicillin manui'actured with corn steep liquor as the principal nutrient source, which would indicate that the associated impurities are either less in the cottonseed meal medium, or that their nature is such that they are rapidly eliminated during the extraction procedure wherein penicillin is extracted from the nutrient broth.

In making up media for growing penicillin-producing microorganisms, such as Penicilllum notatum or Penicillium chrysogenum, in accordance with our invention, an aqueous medium containing cottonseed meal and a carbohydrate, together with various salts such as sodium nitrate, calcium carbonate, ammonium phosphate, magvarious commercially available varieties of been found equally satisfactory. Cottonseed meal, either before or after removal of the cot tonseed. oil, has been found equally-satisfactory.

In general, concentrations of the cottonseed meal in the nutrient medium ranging from 0.5% to 6% have been found effective. 1

Among supplementary sources of carbon and energy dextrose, lactose and brown sugar are all satisfactory carbohydrates. A supplementary nitrogen source, such as sodium nitrate, may also be included in the nutrient medium. Theconcentration of sugar present in the medium may be varied over rather wide limits such, for example, as from 0.25% to 5% by weight, based on the weight of the medium. The starch which is present in cottonseed meal and also added starch may also serve as a readily available energy source for the microorganism. The natural content of min.- erals in the cottonseed meal is usually suflicient for growth and penicillin production, thus permitting the elimination, if desired, of the added nutrient salts of that type disclosed, for example, in our previously referred to copending patent applica-' tion.

Moreover, it is not essential to utilize in the nutrient medium special stimulating chemicals of the phenylacetyl derivative type (Pratt and Dufrenoy, 1945). The stimulating efiects of derivatives of this sort such, for example, as phenylacetylethanolamine, is much less in cottonseed meal medium than it is in other media now utilized commercially such as those containing corn steep liquor. Apparently corn steep liquor is deficient in factors tending to permit the production of penicillin, and phenylacetyl derivatives or other stimulating agents are necessary in order to bring the level of penicillin production in menesium sulfate, zinc sulfate, etc., is prepared.

, This is inoculated with a culture of the penicillinproducing organism which may be a specific strain of Penicillium such as Penicillium notatum NRRL 832; Penicillium chrysoaenum Dem'ereo X1612; or Penicillium chrysogenum Wisconsin Q176 (Backus andStaufler 1945). The microorganism is grown in the cottonseed meal broth dia containing corn steep liquor up to that customarily secured in cottonseed meal media without the use of special stimulating chemicals.

As examples'of our improved process for the 7 preparation of penicillin by the growth of penicillin-producing organisms, and of media containing cottonseed meal for use in propagating the microorganisms under conditions wherein penicillin is produced in high yields, the following may be given. These examples give a number of details about specific nutrient media that may be employed, and they are to be regarded as illustrative of our invention and as not necessarily restrictive thereof.

Example 1 The superiority of nutrient media containing.

having the\compositions noted in the table, One

of these nutrient media, containing corn steep solids, also contained a phenylacetyl derivative as an adiuvant in the amount of 0.5 gramper liter.

Nutrient media were prepared, in accordance with our invention, containing cottonseed meal, these media having the compositions noted in the table. It will be observed that one of these media also contained a phenylacetyl derivative as adjuvant, in the amount of 0.5 gram per liter. The media were separately inoculated both days, and '6 daysQin each case, the penicillin potency was determined by bioassay. The values of penicillin potency given in the table are in Oxford units per milliliter. It will be observed that the penicillin potency wasmuch higher when the microorganism was propagated in nutrient media containing cottonseed meal, as compared with the usual commercially used nutrient medium containing corn steep solids.

dium.

Calcium carbonate 1% I Cottonseed meal 4% Lactose3% Phenylacetylethanolamine I 0.05

Castor oil (antifoam agent) 100 milliliters Tap water balance V The bat-ch was sterilized for /2 hour at 120 C. After cooling to 25 C. it was inoculated with 30 liters of vegetative growth of Penicillium chrysby weight, based on the total weight of the me-- ogenum X1612 from a 48-hour ,old batch which P. chryaogenu'm P. chrumgmum Grams Demerec X1612 Wisconsin Q.176 Medium per liter I 3 days 4 days 5 days days 3 days 4 days days 6 days Corn steep solids 20 Lact 30 140 180 200 120 375 550 650 600 Calcium carbonate 10 a m steep-solids. g8

actose Calcium carbonat 10 160 250 350 300 1, 050 1,300 1, 260 900 Phenylacctyl denva 0.5 Cottonseed meal 40 Lactose 30 190 250 300 175 520 300 950 1, 000 Calcium carbonate.. l0 7 gotgonseed meal. g3

ac ose q I Calcium carbonat 10 160 -70 320 200 540 950 l, 160 885 Phenylacetyl derivatives 0.5

It might be noted that a nutrient medium containing 2% of corn steepsolids was found, by experiment to give the optimum results in penicillin production when corn steep liquor was utilized as the principal constituent of the nutrient medium in which the microorganism was propagated.

Example 2 The batch was first sterilized by heating at 125 C. for hour.- It was then cooled to C. and inoculated with liters of vegetative growth from a 48-hour old batch of Pem'ciZlium chrysogenum X1612 which had been cultivated on the same medium. The medium was continuously agitated and aerated by a propeller at 210 R. P. M. During the propagation period 1000C. F. H. of sterile air was blown through the growing mold and medium. I

Samples of the broth were withdrawn periodically under aseptic conditions and assayed to determine their penicillin content. The maximum activity was found to be 381 Oxford units per milliliter after 56 hours growth. This is an average rate of 6.8 units of penicillin per milliliter of broth per hour of growth.

Example 3 hour of sterile air.

-300 liters of a nutrient medium having the-- following composition was made up in a 100 gallon carbon steel fermenter. The percentages are tics.

Samples were withdrawn periodically under aseptic conditions and assayed to determine their penicillin content. The maximum activity was 293 Oxford units per milliliter after 56 hours growth. This is an average rate of 5.2 units of penicillin per milliliter of broth per hour of growth.

' Example 4 300 liters of medium having the following composition was made up in a gallon carbon steel iermenter. Allpercentages are by weight, based on the total weight of the medium:

Corn steep liquor 4% of solids Lactose 3% Calciumcarbonate 1% Phenylacetylethanolamine 0.05% Y Castor oil (antifoam agent) 100 milliliters Tap water balance The batch was sterilized for k hour at C. After cooling to 25 0.,thebatch was inoculated with 30 liters of vegetative growth of Penicillium chrysogenum X1612 from a 48-hour old batch cultivated on the same medium. The medium was continuously agitated at R. P. M. and aerated with 1000 cubic feet per hour of sterile air. Samples of the nutrient medium were withdrawn periodically under aseptic conditions and assayed for penicillin content. The maximum activity was found to be Oxford units per milliliter of nutrient medium at 106 hours growth. This is an average rate of 1.7 units of penicillin per milliliter per hour.

As a commercial product for use in the manufacture of penicillin by the growth of Penicillia producing penicillin, cottonseed meal had the advantage of uniformity. as contrasted with other media which vary greatly in their characteris- Moreover cottonseed meal is a dry bulk material and is more easily handled than the from the group consistingoi' sodiumnitr'ate, caltype of nutrient media which is supplied commercially in the form of thick viscous syrups.

'-Various changes and modifications might be made in our inventionas described without de-* parting from the scope thereof. Accordingly, to the extent that these changes and modifications are within the scope or the appended claims, they are-to be considered as part or our invention.

We claim: 1. The process for the production of penicillin which comprises propagating a penicillin-producing strain of a mold belonging to the genus Peni cillium in a nutrient medium containing cottonseed meal and a source of carbon and energy selected from the group consisting oi carbohydrates, glycerol,. and mixtures thereof.

3. The process for the production of penicillin which comprises propagating a penicillin-producing strain or a mold belonging to the genus Penicillium in an aqueous nutrient medium comprising cottonseed meal, a source of carbon and energyselected from the group consisting of carbo hydrates, glycerol, and mixtures thereof, and an inorganic salt selected from the group consisting cium carbonate, ammonium phosphate, magnesium sulfate, zinc sulfate, and mixtures thereof.

7. The process'for the production of penicillin which comprises propagating a penicillin-producing strain of Penicillium chrysoaenum in an aqueous medium containing cottonseed meal under aerobic submerged conditions, the pH of said aqueous medium, during said propagation, falling within the range 6.0 to 8.5.

8. The process for theproduction of penicillin which comprises propagating a penicillin-producing strain of Penicillium chrysogenum in a nutrient medium containin cottonseed meal and a source of carbon and energy selected from the group consisting of carbohydrates, glycerol, and

mixtures thereof.

9. The process for the production of penicillin which comprises propagating a penicillin-producing strain of Penicillium chrysoaenum in an aqueous nutrient medium comprising cottonseed meal, a source of carbon and energy selected from the group consisting of carbohydrates, glycerol,

- of sodium nitrate, calciumcarbonate, ammonium phosphate, magnesium sulfate, zinc sulfate, and

mixtures thereof.

4. The process for the production of penicillin which comprises propagating a penicillin-producing strain of Penicillium notatum in an aqueous medium containing cottonseed meal under aerobic submerged conditions, the pH of said aqueous medium, during said propagation, falling within the range 6.0. to 8.5.

5. The process for the production of penicillin which comprises propagating a penicillin-producing strain of Penicillium notatum in a nutrient medium containing cottonseed meal and a source of carbon and energy selected from the group 'COl'lSiSlllIlg of carbohydrates, glycerol, and mixtures thereof.

6. The process for the'production of penicillin which comprises propagating a penicil1in-producing strain of Penicillium notatum in an aqueous nutrient medium comprising cottonseed meal, a source of carbon and energy selected from the group consisting of carbohydrates, glycerol, and mixtures thereof, and an inorganic salt selected and mixtures thereof, and an inorganic salt selected {rpm the group consisting of sodium nitrate, calcium carbonate, ammonium phosphate,

magnesium sulfate, zinc sulfate, and mixtures thereof.

which comprises propagating a penicillin-produm ing'strain of a mold belonging to the genus Penicillium in an aqueous medium containing cotton seed meal, lactose, sodium nitrate, castor oil, and phenylacetyl ethanolamine; under aerobic submerged conditions, the pH of said aqueous medium,.during said propagation, falling within the range 6.0 to 8.5.

11. The process for the production of penicillin which comprises propagating a penicillin-producing strain of a mold belonging to the genus Penicillium in an aqueous medium containing cottonseed meal, lactose, calcium carbonate, castor oil, and phenylacetyl ethanolamine, under aerobic submerged conditions, the pH at said aqueous medium, during said propagation, talling within the range 6.0,to 8.5.

12. The process for the production of penicillin which comprises propagating a penicillin-producing strain of a mold belonging to the genus Penicillium in .an aqueous medium containing 

